A translocation signal for delivery of oomycete effector proteins into host plant cells

Article metrics

Abstract

Bacterial1, oomycete2 and fungal3 plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system1, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors2. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein4,5. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK—representing a change that conserves physicochemical properties of the protein—P. infestans fails to deliver Avr3a or an Avr3a–GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Recognition of Avr3a by R3a follows delivery into the host cell by the T3SS of Pectobacterium atrosepticum (Pba).
Figure 2: Replacement of RXLR-EER motifs with alanine residues, singly or in combination, or with amino acids KMIK-DDK, prevents delivery of Avr3a into host cells.
Figure 3: Avr3a is secreted from haustoria and is translocated into the host cell in an RXLR-EER-dependent manner.

References

  1. 1

    Alfano, J. R. & Collmer, A. Type III secretion system effector proteins: double agents in bacterial disease and plant defense. Annu. Rev. Phytopathol. 42, 385–414 (2004)

  2. 2

    Birch, P. R. J., Rehmany, A. P., Pritchard, L., Kamoun, S. & Beynon, J. L. Trafficking arms: oomycete effectors enter host plant cells. Trends Microbiol. 14, 8–11 (2006)

  3. 3

    Ellis, J., Catanzariti, A. M. & Dodds, P. The problem of how fungal and oomycete avirulence proteins enter plant cells. Trends Plant Sci. 11, 61–63 (2006)

  4. 4

    Armstrong, M. R. et al. An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognised in the host cytoplasm. Proc. Natl Acad. Sci. USA 102, 7766–7771 (2005)

  5. 5

    Bos, J. I. et al. The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana . Plant J. 48, 165–176 (2006)

  6. 6

    Jia, J., Adams, S. A., Bryan, G. T., Hershey, H. P. & Valent, B. Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. EMBO J. 19, 4004–4014 (2000)

  7. 7

    Dodds, P. N., Lawrence, G. J., Catanzariti, A. M., Ayliffe, M. A. & Ellis, J. G. The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells. Plant Cell 16, 755–768 (2004)

  8. 8

    Ridout, C. J. et al. Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance. Plant Cell 18, 2402–2414 (2006)

  9. 9

    Allen, R. L. et al. Host–parasite co-evolutionary conflict between Arabidopsis and downy mildew. Science 306, 1957–1960 (2004)

  10. 10

    Shan, W. et al. The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b . Mol. Plant Microbe Interact. 17, 394–403 (2004)

  11. 11

    Rehmany, A. P. et al. Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines. Plant Cell 17, 1839–1850 (2005)

  12. 12

    Hiller, N. L. et al. A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science 306, 1934–1937 (2004)

  13. 13

    Marti, M., Good, R. T., Rug, M., Kuepfer, E. & Cowman, A. F. Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306, 1930–1933 (2004)

  14. 14

    Bhattacharjee, S. et al. The malarial host-targeting signal is conserved in the Irish potato famine pathogen. PLoS Pathogens 2 e50 doi: 10.1371/journal.ppat.0020050 (2006)

  15. 15

    Haldar, K., Kamoun, S., Hiller, N. L., Bhattacharjee, S. & van Ooij, C. Common infection strategies of pathogenic eukaryotes. Nature Rev. Microbiol. 4, 922–931 (2006)

  16. 16

    Tyler, B. M. et al. Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313, 1261–1266 (2006)

  17. 17

    Toth, I. K. & Birch, P. R. J. Rotting softly and stealthily. Curr. Opin. Plant Biol. 8, 424–429 (2005)

  18. 18

    Holeva, M. C. et al. Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp. atroseptica putative Type III secreted effector (DspE/A) and helper (HrpN) proteins. Mol. Plant Microbe Interact. 17, 943–950 (2004)

  19. 19

    Denecke, J., Botterman, J. & Deblair, R. Protein secretion in plant cells can occur via a default pathway. Plant Cell 2, 51–59 (1990)

  20. 20

    Lindeberg, M. et al. Closing the circle on the discovery of genes encoding Hrp regulon members and type III secretion system effectors in the genomes of three model Pseudomonas syringae strains. Mol. Plant Microbe Interact. 19, 1151–1158 (2006)

  21. 21

    Grenville-Briggs, L. J. et al. Elevated amino acid biosynthesis in Phytophthora infestans during appressorium formation and potato infection. Fungal Genet. Biol. 42, 244–256 (2005)

  22. 22

    Avrova, A. O., Venter, E., Birch, P. R. J. & Whisson, S. C. Profiling and quantifying differential gene transcription in Phytophthora infestans prior to and during the early stages of potato infection. Fungal Genet. Biol. 40, 4–14 (2003)

  23. 23

    Torto, T. A. et al. EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. . Genom. Res. 13, 1675–1685 (2003)

  24. 24

    Blanco, F. A. & Judelson, H. S. A bZIP transcription factor from Phytophthora interacts with a protein kinase and is required for zoospore motility and plant infection. Mol. Microbiol. 56, 638–648 (2005)

  25. 25

    van West, P. et al. Green fluorescent protein (GFP) as a reporter gene for the plant pathogenic oomycete Phytophthora palmivora . FEMS Microbiol. Lett. 178, 71–80 (1999)

  26. 26

    Judelson, H. S., Tyler, B. M. & Michelmore, R. W. Transformation of the oomycete pathogen, Phytophthora infestans . Mol. Plant Microbe Interact. 4, 602–607 (1991)

  27. 27

    Guttman, D. S. et al. A functional screen for the Type III (Hrp) secretome of the plant pathogen Pseudomonas syringae . Science 295, 1722–1726 (2002)

Download references

Acknowledgements

This work was supported by grants from the Scottish Executive Environment and Rural Affairs Department (SEERAD) to S.C.W., P.R.J.B. and I.K.T. J.G.M. is supported by the Universidad Nacional de Colombia sede Palmira Agricultural Sciences department and the European Union Alban Programme. P.v.W. is supported by The Royal Society; L.M. is supported by a Commonwealth Scholarship and Fellowship Plan. The authors thank G. Cowan, H. Liu and E. Venter for technical assistance.

Author Contributions S.C.W., P.R.J.B., P.v.W. and I.K.T developed the concept and designed experiments. S.C.W. and S.G. performed P. infestans transformations and plant inoculations. P.C.B. carried out confocal microscopy and advised on cell biology. S.C.W. performed GUS assays and light microscopy. A.O.A. and J.G.M. quantified gene expression. Antibody detection of tagged transformants was performed by I.H. and S.C. L.M., J.G.M., E.M.G. and M.R.A. carried out experiments with P. atrosepticum. L.P. conducted all bioinformatics analyses.

Author information

Correspondence to Stephen C. Whisson or Paul R. J. Birch.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints.

Supplementary information

Supplementary Information

The file contains Supplementary Figures S1 – S7 and Legends; Supplementary Methods and Supplementary Tables S1 – S5. Figure S1 summarizes the main findings of the paper. Figure S2 and Table S1 show gene expression profiles for P. infestans RxLR-EER genes, and figures S3-S7 and tables S2-S3 support RxLR-EER mediated translocation of Avr3a inside plant cells. (PDF 2025 kb)

Supplementary Information

The file contains Supplementary Bioinformatics Analyses: Identification of candidate RXLR proteins in the Phytophthora infestans draft genome sequence, and in the P. sojae and P. ramorum genome sequences, and design of conservative RXLR motif substitutions. Hidden Markov Model and Heuristic predictions of RxLR-EER proteins from oomycete genomes. (PDF 1186 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Whisson, S., Boevink, P., Moleleki, L. et al. A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450, 115–118 (2007) doi:10.1038/nature06203

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.